QR 

& 


Studies  on  the  Locus  of 
Antibody  Formation 


1913 


-NRLF 


IV 

T3>- 


STUDIES  ON  THE  LOCUS  OF  ANTIBODY 
FORMATION 


BY 


FREDERICK  P.  GAY  AND  G.  Y.  RUSK 

From  the  Hearst  Laboratory  of  Pathology  and  Bacteriology 
University  of  California 


Reprinted  from  the  Transactions  of  the  Fifteenth  International 

Congress  on  Hygiene  and   Demography,  held  at 

Washington,  D.  €.,  September  23-28,  1912 


(10)  WASHINGTON  :  GOVERNMENT  PRINTING    OFFICE  :  1913 


• 


STUDIES  ON  THE  LOCUS  OF  ANTIBODY  FORMATION. 

FREDERICK  P.  GAY  and  G.  Y.  RUSK,  from  the  Hearst  Laboratory  of  Pathology 
and  Bacteriology,  University  of  California. 

Since  the  critical  work  of  Knorr 1  on  toxins  it  has  been  generally 
accepted  that  antibodies  are  formed,  not  by  a  simple  inversion  of 
antigens,  but  by  a  reaction  on  the  part  of  the  cells  of  the  animal  that 
has  received  the  antigen.  Correlatively,  it  has  been  assumed  that  cer- 
tain cells  have  a  particular  affinity  for  a  given  antigenic  substance 
and  are  presumably  specifically  fitted  to  produce  the  corresponding 
antibody.  Ehrlich's  receptor  hypothesis,  while  stating  this  assump- 
tion more  concretely,  has  in  no  instance  given  direct  proof  that  any 
particular  type  of  cell  gives  rise  to  any  given  antibody.  The  experi- 
ment of  Wassermann  and  Takaki 2  that  demonstrated  the  apparent 
neutralization  of  tetanus  toxin  by  brain  substance  is  no  longer  re- 
garded as  a  proof  of  the  nerve-cell  origin  of  tetanus  antitoxin.  In- 
deed the  work  of  Loewi  and  Meyer 3-  would  show  that  injection  of 
the  toxin  into  nervous  tissue  produces  an  increased  susceptibility  of 
the  animal  to  tetanus  toxin  rather  than  an  increased  resistance. 
The  fact  that  tetanus  toxin  disappears  rapidly  from  the  circulating 
blood  of  susceptible  animals  and  may  soon  be  demonstrated  in  the 
central  nervous  system  would  not,  it  would  seem,  prove  conclusively 
that  the  toxin  may  not  also  have  been  fixed  and  neutralized  by  other 
body  cells.  At  least  it  would  seem  necessary  to  assume  that  the 
cells  responsible  for  the  antitoxin  formation  must  first  fix  the  an- 
tigen. This  proof  of  antigen  fixation,  indeed,  constitutes  one  of  the 
methods  that  have  been  employed  in  searching  for  the  locus  of  an- 
tibody formation.  The  only  other  apparent  method  of  determining 
antibody  origin  would  seem  to  lie  in  the  early  demonstration  of 
antibodies  in  given  cell  groups  before  they  are  demonstrable  in  the 
circulating  blood. 

Very  little  information,  therefore,  on  the  site  of  antibody  forma- 
tion has  been  gained  from  studies  on  toxin  and  antitoxin.  Our 
information,  inconclusive  as  it  is,  has  been  obtained  from  the  work 
with  other  antibodies,  and  we  would  do  well  to  consider  first  what 
data  have  been  accumulated  in  respect  to  each  of  the  antibody 

272713 


2  SECTION  I.    HYGIENIC   MICROBIOLOGY  AND  PARASITOLOGY. 

RACTERIOLYSINS. 

Pfeiffer  and  Marx4  inaugurated  the  first  systematic  attempts  to 
discover  the  origin  of  lytic  antibodies.  Their  work  would  seem  to 
indicate  very  clearly  that  the  protective  antibodies  directed  against 
the  cholera  spirillum  are  elaborated  in  the  leucopoietic  organs,  par- 
ticularly in  the  spleen,  but  to  a  less  extent  in  the  bone  marrow,  inas- 
much as  extracts  of  these  organs  protect  guinea  pigs  from  infection 
before  the  blood  serum  does.  Deutsch5  essentially  corroborated 
these  findings  with  B.  typhosus  and  Castellani 6  with  B.  dysenteriae. 
Levaditi's7  conclusions  from  his  work  with  the  spirillum  of  hen 
septicemia  were  likewise  corroborative,  but  his  conclusions  would 
not  seem  justified  by  the  experimental  evidence.  These  authors  all 
agree  that  the  spleen  is  not  essential,  as  its  removal  at  best  but 
slightly  inhibits  antibody  formation;  the  bone  marrow  and  lymph 
nodes  are  secondarily  concerned. 

A  careful  inspection  of  Levaditi's  experiments  would  seem  to  point 
to  the  blood  stream  as  a  possible  source  of  antibodies,  although  his 
conclusions  are  different.  Several  authors,  however,  have  attempted 
to  disprove  experimentally  the  local  or  leucocytic  origin  of  the  bac- 
teriolysins.  Thus  Stenstrom8  found  that  the  injection  of  bacteria 
plus  leucocytes  led  to  less  antibody  production  than  the  injection  of 
bacteria  alone.  Pfeiffer  and  Marx  found  less  antibodies  in  the 
ground  leucocytes  of  immunized  animals  than  in  the  plasma. 
Deutsch  found  the  lysins  were  not  present  in  peritoneal  exudate  and 
Paetsch8  extended  this  finding  so  as  to  include  both  pleural  and 
peritoneal  exudates  and  the  lining  endothelia  of  these  cavities. 

Violle's 10  injections  into  the  gall  bladder  for  the  rapid  production 
of  antibodies  indicate  the  possible  function  of  the  liver  in  this  con- 
nection, which  is  emphasized  more  distinctly  in  work  with  other 
antibodies. 

HEMOLYSINS. 

Metchnikoff11  and  Cantacuzene 12  originally  suggested,  on  what 
would  seem  to  be  largely  philosophical  grounds,  that  the  hemolysins 
may  be  formed  by  the  leucocytes  owing  to  the  recognized  phagocy- 
tosis of  blood  cells  by  the  mononuclears.  The  output  of  "  fixatives," 
according  to  Metchnikoff,  varies  directly  with  the  degree  of  phago- 
cytosis. There  is  direct  evidence  that  goes  to  prove,  however,  that 
these  bodies  are  not  formed  in  the  blood  stream.  McGowan 1S 
showed  that  no  leucocytosis  follows  the  injection  of  alien  blood  and 
Hektoen  and  Carlson 14  have  shown  by  transfusion  experiments  that 
the  antigenic  properties  of  foreign  blood  cells  disappear  from  the 
circulation  within  seven  hours. 


Gay  and  Rusk.]  THE    LOCUS   OF   ANTIBODY   FORMATION.  3 

Among  the  fixed  tissues,  the  liver  and  spleen  seem  to  have  shared 
the  honors  as  the  possible  sites  of  hemolysin  formation.  Leuck- 
hardt  and  Becht,13  following  the  work  of  Hektoen  and  Carlson, 
found  that  the  spleen  alone  of  the  organs  in  a  dog  that  has  received 
goat  or  rat  corpuscles  24  hours  previously  has  the  property  of  im- 
munizing new  animals.  As  a  proof  of  the  temporary  location  of  the 
red  blood  cells  that  have  been  injected,  this  evidence  is  undoubted, 
although  well  recognized  from  other  work;  as  a  proof  of  the  spleen 
as  the  site  of  antibody  formation  it  would  seem  to  be  negative.  The 
statement  by  London16  that  splenectomy  decreases  the  formation  of 
hemolysins  is  categorically  denied  by  Jakusche witch.17  Brezina  18 
found  that  a  specific  serum  against  the  leucopoietic  organs  had  no 
effect  in  disturbing  hemolysin  formation.  Carrel  and  Ingebristen 19 
have  produced  hemolysins  in  the  growing  embryonic  spleen. 

The  evidence  in  favor  of  the  liver  as  the  site  of  hemolysin  for- 
mation is  more  positive.  Both  Cantacuzene  12  and  McGowan  13  have 
shown  the  function  of  Kiipfers  cells  in  the  destruction  of  red  blood 
corpuscles.  Muller,20  in  an  interesting  paper,  has  apparently  traced 
normal  hemolysin  formation  to  the  liver  and  has  even  been  able  to 
stimulate  its  excretion  in  the  liver  suspended  in  Ringer's  solution 
outside  the»body,  by  transfusing  it  with  solutions  containing  iodine 
(iodipin).  A  further  contribution  to  the  stimulating  effect  of  iodin 
compounds  is  given  by  Hektoen 21  who  was  able  to  increase  the  output 
of  hemolytic  sensitizers  in  dogs  by  injecting  sodium  iodoxybenzoate. 
Violles  10  method  of  producing  antibodies  by  gall-bladder  injection 
has  already  been  referred  to  under  bacteriolysins. 

AGGLUTININS. 

Whereas  the  evidence  for  bacteriolysin  formation  seemed  to  favor 
formation  in  the  spleen  or  liver,  the  evidence  for  the  locus  of  origin 
of  the  bacterial  agglutinins  points  distinctly  to  the  blood  stream. 
Thus  the  work  of  Deutsch,5  Castellani,6  Rath,22  Weil  and  Braun,23 
and  Kraus  and  Schiffrnann  24  all  shows  that  the  agglutinins  appear 
in  the  blood  serum  before  they  are  present  in  the  extract  of  any 
organ.  Although  Gruber25  originally  suggested  that  the  polymor- 
phonuclears  form  the  agglutinins,  no  experimental  evidence  goes  to 
prove  this.  The  experiments  of  Achard  and  Bensaud,26  Widal  and 
Sicard,27  of  Paetsch,9  and  of  Kraus  and  Schiffmann24  all  seem  to 
disprove  leucocj^tic  or  local  origin. 

There  is  some  evidence  of  agglutinin  formation  in  the  spleen 
offered  by  v.  Emden,28  Jatta,29  and  Girgoleff.30 

PRECIPITINS  (ANTIBODIES  OF  SOLUBLE  ANTIGENS). 

In  the  case  of  precipitin  formation,  again  the  evidence  seems 
divided.  It  is  shown  by  two  sets  of  observers  (Petit  and  Carlson,81 


4  SECTION   I.    HYGIENIC    MICROBIOLOGY  AND   PARASITOLOGY. 

Vaughan,  Gumming,  and  McGlumphy32)  that  soluble  antigenic  sub- 
stances like  egg  white  or  serum  apparently  disappear  within  a  few 
hours  from  the  circulating  blood.  This  is  shown  by  the  impossi- 
bility of  producing,  in  the  one  set  of  experiments,  antibodies  in 
another  animal  that  is  liberally  transfused  with  such  blood,  and, 
on  the  other  hand,  by  the  failure  to  produce  anaphylaxis  in  guinea 
pigs  to  the  substance  originally  injected  (egg  white,  Vaughan  and 
collaborators).  In  apparent  contradiction  are  the  observations  of 
several  observers  on  the  relation  of  leucocytosis  to  precipitin  forma- 
tion. Thus  both  Cantacuzene 83  and  Swerew 34  have  noted  a  marked 
hyperleucocytosis  preceded  by  an  absolute  decrease  in  polymor- 
phonuclears,  which  may  reasonably  be  related  to  the  liberation  of 
'precipitins.  This  observation  fits  in  neatly  with  that  of  Hiss  and 
Zinsser,35  who  obtained  nonspecific  bacterial  precipitins  from  leuco- 
cytic  extracts,  and  that  of  Stenstrom.8  who  found  that  hemologous 
leucocytes  injected  with  the  precipitinogen  increases  precipitin  out- 
put. Kraus  and  Schiffmann 24  emphatically  regard  the  blood  as  the 
source  of  precipitins,  whereas  Cantacuzene.  in  spite  of  his  evidence 
in  favor  of  the  leucocytes,  is  inclined  to  trace  precipitin  formation 
to  the  spleen. 

The  liver  is  by  no  means  to  be  overlooked  in  discussing  precipitin 
formation.  The  work  of  Manwaring,36  of  Nolf,37  and  of  Balizot,88 
on  anaph}rlactic  shock,  would  seem  to  point  to  the  liver  as  the  seat 
of  action,  and  so,  indirectly,  owing  to  the  relation  that  exists  between 
anaphylaxis  and  precipitins,  as  a  possible  location  of  the  latter 
substances. 

It  is  evident  from  this  brief  survey  of  the  literature  that  no  general 
statement  can  be  made  on  the  locus  of  antibody  formation  in  gen- 
eral. It  may  well  be  that  each  of  the  antibody  types  is  produced 
in  a  different  place  or  places.  But  even  when  we  consider  the  pos- 
sible seat  of  origin  of  any  particular  class  of  antibodies,  we  are 
struck  by  the  apparent  confusion  in  the  acquired  data.  In  the  case 
of  any  of  them  we  may  still  say  that  the  antibody  may  be  formed 
either  in  the  blood  stream  or  in  the  fixed  tissues.  There  seems 
greatest  agreement  on  the  point  that  antibodies  are  formed  either 
by  the  leucocytes  or  the  leucocyte- forming  organs.  And  yet  a  good 
deal  of  recent  work  points  with  increasing  emphasis  to  the  liver,  an 
organ  which,  in  view  of  its  other  functions,  might  logically  likewise 
serve  to  produce  antibodies. 

Our  own  studies  on  antibody  formation  have  been  actively  in 
progress  for  over  a  year.  We  regard  them  hitherto  as  largely  pre- 
liminary and  they  have  led  rather  toward  establishing  certain  meth- 
ods of  attack  and  the  evolution  of  working  hypotheses  of  possible 
heuristic  value  than  to  any  conclusion  on  the  main  subject  at  issue. 


Gay  and  Rusk.]  THE    LOCUS    OF   ANTIBODY   FORMATION.  5 

Certain  by-products  of  the  investigation  are  in  themselves  of  distinct 
interest,  although  their  results  are  negative  in  so  far  as  explaining 
antibody  formation  is  concerned.  Two  incidental  investigations 
may  first  be  summarized  before  dealing  with  the  work  that  bears 
more  directly  on  the  site  of  antibody  formation : 

I.    THE  EFFECT  OF  IODIPIN  ON  THE  OUTPUT  OF  ARTIFICIAL  HEMOLYSINS.38 

Muller's20  experiments  led  him  to  the  conclusion  that  both  nor- 
mal sensitizer  (amboceptor)  and  alexin  are  formed  in  the  liver, 
but  that  their  output  depends  on  the  stimulating  action  of  the 
iodin  of  the  thyroid  gland.  This  author  finds  that  the  injection  of 
thyroid  preparations,  or  of  various  iodine  preparations,  notably  of 
iodipin  (Merck,  25  or  10  per  cent  of  iodin),  produces  a  distinct 
increase  in  from  24  to  36  hours  of  the  normal  hemolysins  in  rab- 
bits and  other  animals.  As  already  stated,  this  increase  comprises 
not  only  an  increase  in  alexin,  as  tested  on  blood  cells  sensitized  by 
an  artificial  hemolysin,  but  an  increase  of  the  normal  hemolytic 
sensitizer.  Hektoen 21  has  further  found  an  increase  in  hemolytic 
sensitizers  in  dogs  that  received  a  single  dose  of  goat's  blood  over 
the  amount  produced  in  control  dogs  on  giving  injections  of  sodium 
iodoxybenzoate.  This  alleged  increase  of  normal  hemolytic  sensi- 
tizers immediately  suggested  the  importance  of  determining  the 
effect  of  iodin  on  artificial  hemolysins,  not  only  as  a  matter  in  itself 
of  theoretical  and  perhaps  of  practical  importance,  but  as  bearing 
on  the  origin  of  antibody  formation.  Differences  that  might  appear 
in  the  hemolytic  potency  of  the  sera  of  immunized  animals  would 
presumably  be  more  striking  than  corresponding  differences  in  nor- 
mal hemolysins,  owing  to  the  much  greater  strength  of  immune  sera. 

Our  experiments  deal,  first,  with  the  effect  of  a  single  injection  of 
iodipin  (Merck,  10  per  cent)  in  rabbits  that  had  been  immunized  by 
several  injections  of  washed  guinea-pig  corpuscles.  In  several  ex- 
periments a  control  animal  that  had  been  immunized  in  a  similar 
manner,  but  that  received  no  iodipin,  was  carried  through.  The 
animals  with  and  without  iodipin  were  bled  before  injection  from  the 
ear  and  at  intervals  subsequently;  the  separate  sera  were  heated  at 
once  to  56°  for  one-half  hour,  and  at  the  end  of  the  experiment  all 
were  tested  for  hemolytic  units  at  the  same  time  with  the  same  cor- 
puscles and  alexin.  In  other  experiments  a  critical  intravenous  re- 
injection  of  the  antigenic  blood  was  given  in  two  highly  immunized 
rabbits,  and  on  the  following  day  one  of  them  was  given  iodipin. 
Both  sera  were  then  tested  at  intervals  for  hemolytic  potency.  There 
was  no  evidence  from  any  of  these  experiments  that  the  injection  of 
iodipin  will  increase  the  output  of  artificial  hemolysins  in  the  im- 
munized rabbits. 


6  SECTION   I.    HYGIENIC   MICKOBIOLOGY   AND   PARASITOLOGY. 

II.  HISTOLOGICAL  STUDIES  OF  THE  TISSUES  IN  IMMUNIZED  ANIMALS  WITH 
A  COMPARISON  OF  CHEMICAL  AND  MICROCHEMICAL  TESTS  FOR  GLYCO- 
GEN.40 

The  histopathological  studies  of  Gay  and  Southard41  in  serum 
anaphylaxis  in  the  guinea  pig  have  seemed  an  interesting  contribu- 
tion to  the  relation  of  structure  to  function.  These  authors  found 
that  anaphylactic  intoxication  is  accompanied  by  definite  lesions  in 
the  nature  of  hemorrhages  and  more  particularly  by  fatty  changes 
in  parenchyma  and  in  the  endothelium.  These  endothelial  fat 
changes  could  be  produced  in  a  few  minutes  following  intravenous 
injection  and  obviously  bear  direct  relation  to  the  cyclonic  symptoms 
of  the  syndrome.  It  occurred  to  us  that  similar  evidence  of  func- 
tional cellular  activity  might  be  histologically  demonstrable  in  the 
cells  that  are  engaged  in  antibody  formation.  For  this  purpose 
rabbits  were  highly  immunized  by  repeated  intravenous  injection  of 
washed  guinea-pig  corpuscles  and  following  a  rest  of  two  or  more 
weeks  were  given  a  critical  reinjection  of  1  cubic  centimeter  of 
washed  guinea-pig  blood  and  at  subsequent  intervals  bled  and  the 
tissues  fixed  in  various  ways  and  stained  by  many  methods.  The 
critical  reinjection  was  aimed,  obviously,  to  obtain  the  antibody- 
producing  cells  in  a  condition  of  highest  activity. 

In  the  first  experiment  a  series  of  immunized  rabbits  received 
each  the  critical  reinjection  of  antigen  and  were  then  bled  at  1,  4. 
and  24  hours  and  4  and  6  days  subsequently.  A  careful  histological 
study  of  tissues  from  this  series  showed  in  the  24-hour  animal  a  very 
marked  increase  of  glycogen  in  the  liver  (Best's  carmine  stain,  alco- 
hol fixation).  The  animals  bled  before  and  after  this  period  gave 
a  regular  increase  and  decrease  of  glycogen  to  the  24-hour  maximum. 
A  more  careful  control  of  this  experiment  has  involved  us  in  techni- 
cal difficulties  that  seemed  too  time  consuming  and  led,  moreover,  to 
far  less  encouraging  results  than  the  first  experiment.  The  question 
of  the  effect  of  starvation  on  the  amount  of  glycogen  alone  seemed 
unsurmountable ;  it  was  found,  for  example,  that  rabbits  that  have 
not  been  fed  for  two  days  have  stomachs  stuffed  with  food.  We  are 
still  in  doubt  as  to  the  significance  of  our  first  find  of  increased 
glycogen.  An  attempt  to  correlate  the  microchemical  reaction  of 
glycogen  with  a  careful  chemical  analysis  of  total  glycogen  in  the 
liver  has,  however,  led  to  results  of  importance.  So  far  as  we  are 
aware,  there  has  been  no  systematic  comparison  between  the  chemical 
analysis  of  an  organ  and  microchemical  staining  reactions  in  a  sam- 
ple of  it.  Dr.  Rusk  has  studied  the  amount  and  distribution  of 
glycogen  in  sections  of  22  rabbit  livers  stained  by  the  Ehrlich  iodin 
method  and  the  Best  carmine  method,  with  a  chemical  analysis  of  a 
greater  (weighed)  portion  of  the  same  livers,  following  Pfluger's 


Gay  and  Rusk.]  THE   LOCUS   OF  ANTIBODY  FORMATION.  7 

method  to  the  conversion  of  glycogen  to  glucose,  and  at  this  point 
introducing  Betrand's  modification  of  Fehling's  method  as  more 
accurate  in  measuring  the  amount  of  copper  reduced.  It  is  found 
that  the  microchemical  method  serves  to  give  somewhat  definite  in- 
formation as  to  the  amount  of  glycogen  present,  but  within  a  limited 
range  only,  for  when  the  chemical  analysis  showed  very  much  or 
very  little  glycogen  the  staining  method  was  at  times  wholly 
inadequate. 

III.    THE   FATE    OF    HORSE    SERUM    INJECTED    INTRAVENOUSLY    IN    NORMAL 
AND  IMMUNIZED  RABBITS. 

The  two  general  methods  that  have  been  and  may  be  employed  in 
seeking  antibody  origin  are  either  to  trace  the  course  of  the  injected 
antigen  to  some  group  of  cells  or  to  seek  the  precocious  appearance 
of  antibodies  in  extracts  of  a  given  group  of  cells.  The  latter  method 
is  the  one  that  has  been  used  most  frequently,  but  it  is  the  former 
that  we  have  employed.  Our  observations,  some  of  which  have 
already  been  published,42  began  with  a  study  of  the  fate  of  horse 
serum  injected  into  the  blood  stream  of  rabbits  that  had  been  im- 
munized against  horse  serum.  They  have  since  led  to  further  studies 
on  the  result  of  an  initial  injection  of  horse  serum  in  normal  rabbits. 
In  all  instances  our  results  deal  with  an  injection  of  one  cubic  centi- 
meter of  serum  intravenously. 

In  beginning  the  experiments  with  immunized  animals  it  was  neces- 
sary first  to  determine  the  best  method  of  detecting  the  antigen  that 
was  reinjected.  It  was  found  that  when  horse  serum  is  injected  intra- 
venously in  rabbits  that  have  a  high  precipitin  content  for  horse  serum 
it  nevertheless  remains  demonstrable  by  the  fixation  reaction  or  the 
precipitin  reaction  for  24  hours.  The  reactions  are  carried  out  by 
adding  the  antigen-containing  antiserum  to  a  pure  antiserum.  After 
48  hours  the  antigen  is  no  longer  demonstrable.  The  persistence  of 
the  antigen  in  the  immune  animal  is  accompanied  by  a  fall  in  the 
precipitin  value  of  serum  (negative  phase).  It  is  of  interest  to  note 
that  although  this  antigen-containing  antiserum  will  not  precipitate 
or  fix  alexin  spontaneously,  it  will  react  with  another  antigenic  anti- 
serum  as  well  as  with  a  pure  antiserum.  It  was  rather  surprising  to 
us  to  fail  in  any  conclusive  demonstration  of  the  antigen  by  the 
fixation  reaction  in  extracts  of  the  organs  of  these  same  immunized 
animals  (spleen,  lymph  nodes,  liver,  kidney,  and  muscles)  either 
at  the  same  time  the  antigen  is  present  in  the  blood  or  even  24  hours 
later. 

Of  undoubtedly  greater  significance  is  the  fact  that  neither  the 
antigen-containing  antiserum,  nor  the  organ  extracts  of  the  same 
animal  will  sensitize  guinea  pigs  to  subsequent  intoxication  by  horse 


8  SECTION  I.    HYGIENIC   MICROBIOLOGY  AND  PAEASITOLOGY. 

serum.  We  compared  the  fixing  values  of  pure  horse  serum  and 
antigenic  antiserum,  and  although  one  fixing  dose  of  the  horse  serum 
will  sensitize  guinea  pigs,  many  fixing  doses  of  the  antigenic  anti- 
serum  fail  to  do  so  (at  least  100).  This  would  explain  the  results 
of  Vaughan,  Gumming,  and  McGlumphy32  who  found  that  egg 
white  apparently  disappears  from  the  circulation  in  a  few  hours 
when  tested  for  by  the  anaphylaxis  reaction.  It  is  perhaps  also  in 
harmony  with  the  work  of  Hektoen  and  Carlson14  and  of  Petit 
and  Carlson  81  who  proved  by  transfusion  that  the  antibody-incit- 
ing factor  in  blood  cells  or  in  serum  leaves  the  circulation  in  a  few 
hours.  We  might  possibly  assume  that  the  factor  in  the  antigen  that 
produces  the  antibody  differs  from  the  one  that  unites  with  it.  (Cfr. 
Bang  and  Forsmann.43 

The  results  of  injecting  horse  serum  into  normal  animals  are  also 
of  interest.  The  horse  serum  is  detectable  by  the  precipitin  and 
fixation  reactions  for  several  days.  It  apparently  does  not  sensitize 
in  large  doses  even  after  24  hours.  The  fixation  and  precipittnogen 
antigen  is  present  not  only  in  the  blood,  but  also  in  the  various  organ 
extracts  (in  this  case  carefully  freed  of  blood)  in  uniform  amounts 
on  the  seventh,  eighth,  ninth,  and  tenth  day.  Of  greatest  importance 
is  the  fact  that  the  corresponding  antibodies  have  begun  to  appear 
in  the  serum  two  or  three  days  before  the  antigen  disappears.  Simi- 
lar facts  have  been  noted  by  Hintze.44  It  is  evident  then  that  not 
all  the  antigen  is  used  up  in  producing  the  antibody.  We  have  to 
imagine  either  that  the  antigen  continues  to  unite  with  the  cell  or 
stimulate  the  cell  for  some  time  after  antibodies  appear,  or  that  the 
antibody  stimulant  differs  from  the  antigen  fraction  that  unites  with 
the  antibody.  This  latter  hypothesis  seems  to  us  for  the  present 
the  most  valuable  for  a  working  basis.  It  explains,  moreover,  our 
own  failure  to  produce  anaphylaxis  (cfr.  also  Vaughan  and  his 
collaborators)  ,  and  also  the  results  of  Hektoen  and  Carlson.  We  are 
continuing  our  work  with  this  as  a  working  hypothesis  and  also 
with  a  possible  further  elucidation  of  the  obscure  phenomenon  of 
anaphylaxis  in  mind. 


Fortschr.  der  Mediz.,  etc.,  vol.  15  (1897),  p.  657. 
'Wassermann  and  Takaki  :  Berlin.  Klin.  Wochen.,  vol.  35  (1898).  p.  5. 
'Loewi  and  Meyer:  Archiv  fur  experiment.  Path.,  vol.  59  (1908),  p.  355. 
4Pfelffer  and  Marx:  Zeit  fiir  Hygiene,  vol.  27  (1898),  p.  272. 
5Deutsch:  Annales  de  1'Inst.  Pasteur,  vol.  13  (1899),  p.  689. 

9  Ca  Stella  ni  :  Zeit.  fur  Hygiene,  vol.  37  (1901),  p.  381. 
TLevaditi:  Annales  de  1'Iust.  Pasteur,  vol.  18  (1904),  p.  511. 
'Stenstrom:  Zeit  fiir  Immunitatsforsch.,  vol.  8  (1911),  p.  483. 
*  Paetsch:  Centralblatt  fiir  Bakt,  Grig.  I,  vol.  60  (1911),  p.  255 

10  Violie  :  Annales  de  1'Inst.  Pasteur,  vol.  26  (1912),  pp.  381,  467. 

u  Metchnikoff  :   L'immunite  dans  les  maladies  infectieuses,  p.  103;   Annales 
de  1'Inst.  Pasteur,  vol.  13   (1899),  p.  737. 
"  Cantacuz£ne  :  Annales  de  1'Inst.  Pasteur,  vol.  16  (1902),  p.  522. 


Gay  and  Rusk.]  THE   LOCUS   OF  ANTIBODY 

"McGowan:  Journal  of  Pathology  and  Bacteriology,  vol.  14  (1909),  379; 
vol.  15  (1911),  262. 

"Hektoen  and  Carlson:  Transact.  Chicago  Path.  Soc.,  vol.  S  (1909),  p.  4; 
Joura.  of  Infect.  Diseases,  vol.  7  (1910),  p.  319. 

15  Leuckhardt  and  Becht:  Transact.  Chicago  Path.  Soc.,  vol.  8  (1911),  p.  202. 

"London:  Archiv  fur  biologische  Wissenschaften  (1901),  p.  328. 

17  Jakuschewitsch :  Zeit.  fur  Hygiene,  vol.  47  (1904),  p.  407. 

18Brezina:  Wein.  Klin  Wochenschr.  (1905),  p.  905. 

19  Carrel  and  Ingebristen:  Journ  A.  M.  A.,  vol.  58  (1911),  p.  477. 

"Muller:  Central,  fur  Bakt,  I.  Abt,  Orig.,  vol.  57  (1911),  p.  577. 

21  Hektoen:  Transact.  Chicago  Path.  Soc.,  vol.  8  (1911),  p.  138. 

22 Rath:  Central,  fiir  Bakt,  vol.  25  (1899),  529. 

23  Weil  and  Braun:  Biochem.  Zeitschr.,  vol.  17  (1910),  p.  337. 

24  Kraus  and  Schiffman:  Aunales  de  1'Inst.  Pasteur,  vol.  20  (1906),  225. 
^Gruber:  Miinch.  mediz.  Wochenschr.  (1897),  Nos.  17  and  18. 

26  Ac-hard  and  Bensaud :  Archiv  fiir  med.  Experiment  (1896),  p.  748. 

27Wk!al  arid  Sicard:  Annales  de  1'Inst.  Pasteur,  vol  11  (1897),  p.  353. 

28  v.  Emden:  Zeit.  far  Hygiene,  vol.  30  (1899),  p.  19. 

"•Jatta:  Zeit.  fur  Hygiene,  vol.  33  (1900),  p.  185. 

30Girgoleff:  Zeit.  fiir  Imniunitatsforsch,  vol.  12   (1912),  p.  401. 

81  Petit  and  Carlson:  Jour.  Infect.  Diseases,  vol.  10  (1912),  p.  43. 

82Vaughan,  Cumming  &  McGlumphy:  Zeit.  fiir  Immunitatsforsch.,  vol.  9 
(1911),  p.  16. 

33  Cantacuzene :  Annales  de  1'Inst.  Pasteur,  vol.  22  (1908),  p.  54. 

34Swerew:  Rusaky  Wratsch.  (1910),  p.  367;  Ref.  Jahresbericht  der  Iminuni- 
tatsforschung,  VI2,  p.  527. 

36  Hiss  and  Zinsser:  Jour.  Med.  Research,  vol.  19  (1908),  p.  399. 

38Manwaring:  Zeitschr.  fiir  Immunitatsforsch.,  vol.  8  (1910),  p.  1. 

3TNolf :  Archives  internat.  de  Physiolog.,  Vol.  X  (1910),  p.  37. 

38Blaizot:  Compt.  rend.  heb.  Soc.  de  Biol.,  vol.  70  (1911),  p.  383. 

38  Gay  &  Rusk:  Univ.  Calif.  Publ.  Path.,  vol.  2,  No.  7  (1912). 

40 Rusk:  Univ.  Calif.  Publ.  Path.,  vol.  2,  No.  9  (1912). 

43  Gay  and  Southard:  Jour.  Med.  Research,  vol.  16  (1907),  p.  143. 

42  Gay  and  Rusk :  Univ.  Calif.  Publ.  Path.,  vol.  2,  No.  6  (1912). 

48  Bang  and  Forssmann:  Centralblatt  fiir  Bakt,  I.  Orig.,  XL  (1905),  p.  151. 

"Hintze:  Zeit  fiir  Immunitatsforsch.,  vol.  6  (1910),  p.  113. 

DISCUSSION. 

Dr.  W.  H.  PARK:  Observations  made  on  the  injection  of  toxins  and 
antigens  into  the  blood  or  into  the  subcutaneous  or  other  tissues  in- 
dicate that  for  many  antibodies  several  varieties  of  cells  take  part. 
The  blood  in  an  animal  highly  immunized  to  diphtheria  toxin  contains 
about  100  times  the  quantity  of  antitoxin  as  the  tissue  fluids.  If  toxin 
is  added  to  antitoxin,  union  slowly  takes  place  and  the  combined  toxin 
is  no  longer  able  to  excite  the  production  of  antitoxin.  When  a  horse 
having  a  strongly  antitoxic  blood  is  injected  intravenously  with  a 
definite  amount  of  toxin  very  little  production  of  antitoxin  takes 
place,  because  most  of  it  is  neutralized  by  the  antitoxin  in  the  blood. 
The  same  amount  injected  in  scattered  spots  subcutaneous!}^  will 
produce  a  large  amount  of  antitoxin.  The  part  absorbed  into  the 
blood  would  meet  the  same  fate  as  that  injected  intravenously.  It 


10         SECTION   1.    HYGIENIC  MICROBIOLOGY  AND  PARASITOLOGY. 

seems,  therefore,  certain  that  some  or  all  of  the  cells  in  the  area  in  the 
subcutaneous  tissues  that  the  toxin  reaches  must  take  part  in  pro- 
ducing antitoxin.  If  toxin  is  injected  through  the  trachea  into  the 
lungs  it  acts  in  the  same  way  as  when  injected  subcutaneously.  I 
have  seen  similar,  though  not  as  definite,  results  with  the  injection 
of  various  bacterial  antigens.  These  facts  seem  to  suggest  that 
more  varied  cells  than  Dr.  Gay  indicates  may  take  part  in  antibody 
formation. 

Dr.  VAUGHN  thinks  that  the  ferment  is  formed  by  different  cells 
according  to  the  sensitizer  used.  He  referred  to  the  work  of  J.  W. 
Vaughan  on  sensitization  to  cancer  proteins  in  which  it  appears 
that  the  ferment  is  formed  in  the  large  mononuclear  leucocytes  and 
that  the  sensitization  is  transitory.  The  fact  that  the  nonpoisonous 
part  sensitizes  may  be  due  to  the  presence  of  a  minute  trace  of  un- 
broken proteins.  However,  this  seems  highly  improbable  because 
*  this  part  does  not  sensitize  to  itself. 

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Syracuse,  X.  y. 
PAT,  JIN.  21.  1908 


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